Results of recent clinical trials have generated much excitement and optimism for the potential benefits of fully human antibodies in the diagnosis and treatment of disease. This success is in part due to the technology of selecting antibodies against novel targets from phage display libraries and also due to improved production platforms.
One IgG molecule comprises two heavy chains and two light chains. The heavy chains consist of (starting from the N-terminus): a variable region, a constant region, a hinge region and two additional constant regions (FIG. 1). The hinge region contains cysteine residues that form disulphide bonds with a second heavy chain to mediate dimerization of the protein. The number of cysteine residues varies depending on the IgG sub-class: IgG1 has 2 cysteines that form disulphide bonds in the hinge. The light chains consist of a variable region and a constant region; residues C-terminal to the constant region form disulphide bonds with residues immediately before the heavy chain hinge regions. Thus the four chains are held together by multiple disulphide bonds as well as other noncovalent interactions between the intimately paired chains.
The structure of an antibody may be defined as distinct domains: the Fc region which mediates effector functions and the F(ab′) region which binds antigen (George and Urch, 2000). The two C-terminal constant domains of the heavy chains make up the Fc region. A F(ab′) fragment comprises the light chain and the variable and first constant region of the heavy chain; a F(ab′)2 fragment comprises two F(ab′) fragments dimerized through the heavy chain hinge region (FIG. 1).
Antibodies are under investigation as therapies for a wide range of clinical problems including organ transplantation, cardiac disease, infectious diseases, cancer, rheumatologic and autoimmune disease, neurologic disorders, respiratory diseases, as well as disorders with organs such as the blood, skin and digestive tract.
One of the major focuses for antibody discovery and development is in the field of cancer imaging and therapy (Carter, 2001). Antibodies may be used as naked molecules or they may be labeled and so used as a magic bullet to deliver a cargo to the tumor (Borrebaeck and Carlsson, 2001; Park and Smolen, 2001). A number of naked antibodies are currently in the clinic. While it is clear that they are able to reduce tumor load in patients, the mechanism by which this occurs is unclear. Classically, these might work by recruiting effector cells (via Fc receptors) or complement to the target cell. More recently, it is becoming apparent that they may also function by binding cell surface proteins and then activating inappropriate signaling pathways or apoptotic signaling pathways, leading to cell death (Tutt et al., 1998).
Antibodies are currently used in the clinic, both as intact IgG molecules and as F(ab′) and F(ab′)2 fragments. When choosing an antibody format, there are several important issues. They should have a high antigen avidity and specificity, be sufficiently small to penetrate tumor tissue and remain in the circulation long enough to localize to tumors. In addition (particularly if they are labeled with a radiolabel or other toxic moiety), they should be cleared from the body at a rate which prevents non-specific toxicity or high background.
F(ab′)2 fragments exhibit a number of benefits over intact IgG related to the above points, which make them attractive for imaging and therapy. First, these molecules have a shorter half-life than an intact IgG, because they are more rapidly removed from the circulation by the kidneys as a result of their lower molecular weight, thus reducing potential toxicity (Behr et al., 1995). Another advantage of the reduced size is that they may penetrate tumor tissue and associated vasculature more readily (Yokota et al, 1992). In this way, more cells of the tumor mass are targeted.
Advantages also exist due to the absence of the Fc region of the molecule. The F(ab′)2 fragment does not induce activation of immune responses, as the Fc region (which binds complement and Fc receptors) is absent. This is of particular relevance in imaging studies where only a snapshot of tumor dispersion and size is required. F(ab′)2 fragments also do not have the problem of non-specific binding to targets through the Fc moiety, reducing background and non-specific labeling. The advantages listed above may also apply in part to F(ab′) fragments. However, F(ab′)2 molecules are bivalent (as are intact IgGs) and so should bind target molecules with higher avidity. F(ab′) fragments are monovalent and, as a result, generally exhibit lower avidities. For these reasons, F(ab′)2 fragments are highly desirable as clinical agents.
While advantages of F(ab′)2 fragments are clear, it has not proven as easy to make F(ab′)2 fragments. Several methods are currently available. The classic method is to make intact IgG, digest it with a protease, such as pepsin, to remove the Fc region of the antibody. Other regions of the molecule may, however, be nicked by the protease (including the antigen-binding region, resulting in the loss of binding capacity of the antigen-binding region) and digestion may not be complete. Further purification is then required to remove the F(ab′)2 fragment from the non-digested antibody, the free Fc domain and the protease.
An alternative method is to make F(ab′) fragments in bacteria and then dimerize the molecules to generate F(ab′)2 molecules (Willuda et al., 2001; Zapata et al., 1995; Humphreys et al., 1998; U.S. Pat. No. 5,648,237). Dimerization may use specific self-associating peptides (which may prove antigenic in vivo), conjugation via chemical cross-linkers or in vitro reduction/oxidation of F(ab′)-hinge fragments. These methods require additional purification steps and may produce unusual molecules (such as two F(ab′) fragments linked “head-to-tail” so that the antigen-binding regions are at opposite ends of the new molecule).
Another method for producing F(ab′)2 fragments is to generate them directly in mammalian cells. While this might appear straightforward, it has been observed that F(ab′) fragments are often produced in preference to F(ab′)2 fragments. One report in which CHO cells were used for IgG4 F(ab′)2 production indicated that F(ab′)2 fragments accounted for only 10% of the protein produced, with F(ab′) fragments accounting for 90% (King et al., 1992; King et al., 1994). The reason for this is unclear. Another important cell line in the production of monoclonal antibodies is SP2/0; an attempt to produce IgG1 F(ab′)2 fragments in this cell line yielded essentially only monovalent products. Addition of an IgG3 hinge, which comprises 11 sulfur bridges instead of the two sulfur bridges present in an IgG1 hinge, resulted in the production of 98% divalent product (Leung et al., 1999). However, increased numbers of sulfur bridges generally decreases production levels of the antibody fragments and it is, therefore, preferable to have fewer sulfur bridges for production on a large scale. A similar picture was seen upon expression of F(ab′)2 fragments in COS cells (De Sutter et al., 1992). Thus, despite these efforts, there is still a need for improved production methods of F(ab′)2 fragments. PER.C6™ is a human cell line and an example of an immortalized primary eukaryotic host cell. It is able to grow in suspension culture in serum-free medium, which, upon transfection with an appropriate expression vector and selection of stable cell lines, is capable of producing recombinant protein in abundance, as disclosed in WO 00/63403. In the '403 application, it has been disclosed that PER.C6™ cells can express intact human IgG, but no specific data have been provided for F(ab′)2 fragments.